Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae)

Using the F2 screen methodology, we estimated the frequency of alleles conferring resistance to the Cry1Ab toxin of Bacillus thuringiensis Berliner in a Philippine population of the stem borer Scirpophaga incertulas (Walker). Evaluation of >450 isofemale lines for survival of F2 larvae on cry1Ab plants did not detect the presence of an allele conferring a high level of resistance. The frequency of such an allele in the sampled population was conservatively estimated to be <3.6 x 10(-3) with 95% confidence and a detection probability of 94%. However, there was evidence of the presence of alleles conferring partial resistance to Cry1Ab. The frequency of alleles for partial resistance was estimated as 4.8 x 10(-3) with a 95% CI between 1.3 x 10(-3) and 1.04 x 10(-2) and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab in the population of S. incertulas sampled is not too high to preclude successful implementation of the high dose/refuge resistance management strategy.     

Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of the European corn borer,Ostrinia nubilalis

Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis ( Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)). We used an F(2) screen on >1,200 isofemale lines of Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80%. In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.     

Evaluating resistance to Bt toxin Cry1Ab by F2 screen in European populations of Ostrinia nubilalis (Lepidoptera: Crambidae)

The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the Cry1Ab toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the Cry1Ab toxin. The cost of F2 screen performed in the four countries varied from U.S. dollars 300 to dollars 1300 per line screened. The major difference in cost was mostly due to a severe loss of univoltine lines during the screen in Germany and Slovakia. In none of the screened lines did we detect alleles conferring resistance to Mon810 maize or to the Cry1Ab toxin. The frequency of resistance alleles were < 1.0 x 10(-3), < 1.6 x 10(-3), < 9.2 x 10(-3), and < 2.6 x 10(-2) in France, Germany, Italy, and Slovakia, with 95% probability, respectively. The average detection probability over all lines was approximately 90%. Making the assumption that European corn borer populations in these countries belong to the same genetic entity, the frequency of alleles conferring resistance to the Cry1Ab produced by the Mon810 maize in western and central Europe was 1.0 x 10(-4), with a 95% confidence interval of 0-3.0 x 10(-4).     

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae)

The refuge plus high-dose strategy for resistance management assumes that the frequency of resistance alleles is low. We used an F2 screen to estimate the frequency of resistance to transgenic corn that produces Bacillus thuringiensis Berliner Cry1Ab toxin (Bt corn) in an Iowa population of European corn borer, Ostrinia nubilalis (Hübner). We also proposed a modification to the statistical analysis of the F2 screen that extends its application for nonuniform prior distributions and for repeated sampling of a single population. Based on a sample of 188 isofemale lines derived from females caught at light traps during the 2nd flight of 1997, we show with 95% confidence that the frequency of resistance to Bt corn was <3.9 x 10(-3) in this Iowa population. These results provide weak evidence that the refuge plus high-dose strategy may be effective for managing resistance in O. nubilalis to Bt corn. Partial resistance to Cry1Ab toxin was found commonly. The 95% CI for the frequency of partial resistance were [8.2 x 10(-4), 9.4 x 10(-3)] for the Iowa population. Variable costs of the method were 14.90 dollars per isofemale line, which was a reduction of 25% compared with our initial estimate.     

Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important lepidopteran pest of cotton (Gossypium spp.) in Australia and the Old World. From 2002, F2 screens were used to examine the frequency of resistance alleles in Australian populations of H. armigera to Bacillus thuringiensis (Bt) CrylAc and Cry2Ab, the two insecticidal proteins present in the transgenic cotton Bollgard II. At that time, Ingard (expressing Cry1Ac) cotton had been grown in Australia for seven seasons, and Bollgard II was about to be commercially released. The principal objective of our study was to determine whether sustained exposure caused an elevated frequency of alleles conferring resistance to Cry1Ac in a species with a track record of evolving resistance to conventional insecticides. No major alleles conferring resistance to Cry1Ac were found. The frequency of resistance alleles for Cry1Ac was <0.0003, with a 95% credibility interval between 0 and 0.0009. In contrast, alleles conferring resistance to Cry2Ab were found at a frequency of 0.0033 (0.0017, 0.0055). The first isolation of this allele was found before the widespread deployment of Bollgard II. For both toxins the experiment-wise detection probability was 94.4%. Our results suggest that alleles conferring resistance to Cry1Ac are rare and that a relatively high baseline frequency of alleles conferring resistance to Cry2Ab existed before the introduction of Bt cotton containing this toxin.     

Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae

Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.     

High Genetic Variability for Resistance to Bacillus thuringiensis Toxins in a Single Population of Diamondback Moth

The long-term benefit of insecticidal products based on Cry toxins, either in sprays or as transgenic crops, is threatened by the development of resistance by target pests. The models used to predict evolution of resistance to Cry toxins most often are monogenic models in which two alleles are used. Moreover, the high-dose/refuge strategy recommended for implementation with transgenic crops relies on the assumption that the resistance allele is recessive. Using selection experiments, we demonstrated the occurrence in a laboratory colony of diamondback moth of two different genes (either allelic or nonallelic) that confer resistance to Cry1Ab. At the concentration tested, resistance was dominant in one selection line and partially recessive in the other. Resistant insects from the two selection lines also differed in their cross-resistance patterns. The diamondback moth colony was derived from a field population from the Philippines, which originally showed a different resistance phenotype. This is the first time that an insect population has been directly shown to carry more than one gene conferring resistance to the same Cry toxin.     

High genetic variability for resistance to Bacillus thuringiensis toxins in a single population of diamondback moth.

The long-term benefit of insecticidal products based on Cry toxins, either in sprays or as transgenic crops, is threatened by the development of resistance by target pests. The models used to predict evolution of resistance to Cry toxins most often are monogenic models in which two alleles are used. Moreover, the high-dose/refuge strategy recommended for implementation with transgenic crops relies on the assumption that the resistance allele is recessive. Using selection experiments, we demonstrated the occurrence in a laboratory colony of diamondback moth of two different genes (either allelic or nonallelic) that confer resistance to Cry1Ab. At the concentration tested, resistance was dominant in one selection line and partially recessive in the other. Resistant insects from the two selection lines also differed in their cross-resistance patterns. The diamondback moth colony was derived from a field population from the Philippines, which originally showed a different resistance phenotype. This is the first time that an insect population has been directly shown to carry more than one gene conferring resistance to the same Cry toxin.     

Susceptibility of Spanish populations of the corn borers Sesamia nonagrioides (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Lepidoptera: Crambidae) to a Bacillus thuringiensis endotoxin

Baseline susceptibility to the Cry1Ab delta-endotoxin from Bacillus thuringiensis (Berliner) was determined for four populations of Sesamia nonagrioides (Lefebvre) and two populations of Ostrinia nubilalis (Hübner) from Spain. This study shows that S. nonagrioides is at least as susceptible as O. nubilalis to B. thuringiensis Cry1Ab protein. We found small differences in susceptibility among the Spanish populations of S. nonagrioides that can be attributed to natural variation, because there are no records of B. thuringiensis products being used on corn crops in Spain. There were no differences in susceptibility to Cry1Ab toxin between the two populations of O. nubilalis.     

Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.     

Susceptibility to the Cry1F toxin of field populations of Sesamia nonagrioides (Lepidoptera: Noctuidae) in Mediterranean maize cultivation regions

Maize hybrids expressing the Cry1F toxin provide efficient control of lepidopteran pests. The Mediterranean corn borer, Sesamia nonagrioides (Lefèvre), is one of the most damaging pests of maize in the Mediterranean basin. In this work we firstly determined the efficacy of maize hybrids expressing the Cry1F toxin (event TC1507) to control neonates of S. nonagrioides. Leaf tissue feeding bioassays revealed that TC1507 maize is highly effective against this pest, and the percentage mortality obtained was comparable to that obtained with a Cry1Ab-expressing maize hybrid (Compa CB, event 176), which is known to be highly efficacious against S. nonagrioides. Secondly, interpopulation variation in the susceptibility to the Cry1F insecticidal protein was established for nine field-collected populations of S. nonagrioides (three Spanish, two French, two Italian, one Greek, and one Turkish). Estimates of the susceptibility of larvae to the Cry1F toxin showed low variability in lethal concentrations and growth inhibition concentrations among field populations. Moreover, no significant differences were found when they were grouped by geographical areas [Western Mediterranean (Spain and France) versus Eastern Mediterranean (Italy, Greece and Turkey)] or by history of exposure to Bt plants (Spanish vs. other populations). Therefore, the minor differences found in field populations can be attributed to natural variation in sensitivity to Cry1F. The importance of establishing baselines of susceptibility for resistance detection is discussed. Future changes in susceptibility of S. nonagrioides populations to Cry1F could be documented based on this baseline data.     

Extended monitoring of resistance to Bacillus thuringiensis Cry1Ab maize in Diatraea saccharalis (Lepidoptera: Crambidae)

The sugarcane borer, Diatraea saccharalis (F.), is a major target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid-Southern region of the United States. During 2007-2009, a total of 986 feral individuals of D. saccharalis were collected from maize fields in six locations of Louisiana and Mississippi and examined for resistance to Cry1Ab maize using F 1/F 2 screens. Major resistance alleles to Cry1Ab maize in the populations sampled from non-Bt maize plants during 2007 and 2008 in Louisiana and 2009 in Mississippi were rare. From a total of 487 individuals collected from three locations in Louisiana in 2007 and 2008, only one individual was identified with major resistance alleles. In addition, no major resistance alleles were detected in 242 individuals collected from three locations in Mississippi in 2009. The frequency of major resistance alleles was estimated to be 0.002 with a 95% CI of 0.00025-0.0057 for the Louisiana populations and < 0.0061, with 95% probability, for the Mississippi populations. The resistance frequency estimated for the Louisiana populations in 2007 and 2008 was not significantly different from those reported previously for populations sampled in 2004-2006. However, among 200 individuals sampled from non-Bt maize plants in 2009 in Louisiana, six individuals were identified to possess major resistance alleles. The estimated major resistance allele frequency for the populations sampled from non-Bt maize plants in 2009 in Louisiana was 0.0176 with a 95% CI of 0.0072 to 0.0328, which was significantly greater than those estimated for the populations collected in 2004-2008. Similarly, the frequency of minor resistance alleles to Cry1Ab maize for the Louisiana populations collected in 2009 was also significantly greater than those estimated for the populations sampled before. In addition, two out of 57 feral individuals collected from Bt maize plants in Louisiana in 2009 were identified to carry major resistance alleles to Cry1Ab maize. Since 2010, transgenic maize expressing pyramided Bt genes has been planted in the US mid-Southern region and by 2011, pyramided Bt maize has replaced Cry1Ab maize as the dominant Bt maize for managing lepidopteran pests including D. saccharalis. The timely switching from single-gene Cry1Ab maize to the pyramided Bt maize should prevent further increases in Cry1Ab resistance allele frequency and thus ensure the continued success of Bt maize for managing D. saccharalis in the region.     

Field measures of western corn rootworm (Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in maize event 59122 and implications for trait durability

Maize, Zea mays L., has been transformed to express the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis strain PS149B1. These two proteins act together as a binary insecticidal protein that is effective against corn rootworm (Coleoptera: Chrysomelidae) species. The design of the resistance management plan to preserve the long-term durability of this trait largely depends on the level of rootworm mortality induced by Cry34/35Ab1 corn rootworm-protected maize (frequently referred to as "dose" in this context). Here, we report on studies that showed Cry34/35Ab1-expressing maize event 59122 caused 99.1 to 99.98% mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae, after adjusting adult emergence numbers for density-dependent mortality. In two of three studies, there was a short delay in time to 50% adult emergence from 59122 maize plots compared with control plots, although emergence was completed at approximately the same time from both types of maize. These data support an expectation that alleles conferring resistance to the Cry34/35Ab1 proteins in western corn rootworm will be functionally nearly completely to completely recessive on 59122 maize and that there is unlikely to be assortative mating of Cry34/35Ab1-resistant and susceptible rootworms. When incorporated into simulation models of rootworm adaptation to transgenic maize, these findings suggest that a 20% refuge is likely to be highly effective at prolonging the durability of 59122 maize.     

Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F₁/F₂ isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring.     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.     

Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.     

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern United States Corn Belt population of European corn borer (Lepidoptera: Crambidae)

The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F2 screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10(-3)) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F2 screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.     

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Genetic and biochemical approach for characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in a field population of the diamondback moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F(4) to F(8)) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F(9) found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F(9). The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F(2) progeny from a backcross of F(1) progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.     

Evolution, ecology and management of resistance in Helicoverpa spp. to Bt cotton in Australia

Prior to the widespread adoption of two-gene Bt cotton (Bollgard II?) in Australia, the frequency of resistance alleles to one of the deployed proteins (Cry2Ab) was at least 0.001 in the pests targeted namely, Helicoverpa armigera and Helicoverpa punctigera. In the 7 years hence, there has been a statistically significant increase in the frequency of alleles conferring Cry2Ab resistance in field populations of H. punctigera. This paper reviews the history of deploying Bt cotton in Australia, the characteristics of the isolated Cry2Ab resistance that likely impact on resistance evolution, aspects of the efficacy of Bollgard IIχ, and the behavioural ecology of Helicoverpa spp. larvae as it pertains to resistance management. It also presents up-to-date frequencies of resistant alleles for H. punctigera and reviews the same information for H. armigera. This is followed by a discussion of current resistance management strategies. The consequences of the imminent release of a third generation product that utilizes the novel vegetative insecticidal protein Vip3A are then considered. The area planted to Bt-crops is anticipated to continue to rise worldwide and many biotechnical companies intend to add Vip3A to existing products; therefore the information reviewed herein for Australia is likely to be pertinent to other situations.